The present invention relates generally to an expression system of soluble glutaminyl cyclase (QC).
Glutaminyl cyclases (QCs) (EC 2.3.2.5.) are acyltransferases responsible for the conversion of the protein N-terminal glutaminyl residue into pyroglutamic acid (pGlu) with the concomitant liberation of ammonia. This cyclization reaction is important during the maturation of numerous neuropeptides and cytokines, such as thyrotropin-releasing hormone (TRH), godadotropin-releasing hormone (GnRH) and monocyte chemotactic protein-2 (MCP-2), in the secretory pathway. The role of pGlu on these bioactive peptides is believed to be in developing the proper conformation of the peptides in order to bind to their targets and/or protecting the peptides from exopeptidase degradation.
In humans, the abberant formation of pGlu may be related to some pathological processes, such as osteoporosis and amyloidotic diseases. The plaque forming peptides, e.g., AβN3(pGlu)-40/42, seem to be directly correlated with the severity and progression of the amyloidotic diseases, such as Alzheimer's disease (AD) and Down's syndrome (DS). The pGlu on these plaque-forming peptides is converted from a glutamyl residue, and contributes to the hydrophobicity and proteinase resistance of these peptides. Such a glutamyl-to-pGlu conversion was demonstrated to be also catalyzed by human QC in vitro [S. Schilling, et al., “Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions,” FEBS Lett. 563, 191-196 (2004)].
To date, although functional human QC has been expressed in yeast and insect cell systems, expression of the protein still encounters problems with insolubility, low recovery and heterogeneity of the protein [S. Schilling, et al., “Heterologous expression and characterization of human glutaminyl cyclase: evidence for a disulfide bond with importance for catalytic activity,” Biochemistry 41, 10849-10857 (2002); R. E. Booth, et al., “Human pituitary glutaminyl cyclase: expression in insect cells and dye affinity purification,” Protein Expression &. Purification 32, 141-146 (2003)].